Activation and inhibition of sirtuins: From bench to bedside.

The sirtuin family comprises seven NAD+-dependent enzymes which catalyze protein lysine deacylation and mono ADP-ribosylation. Sirtuins act as central regulators of genomic stability and gene expression and control key processes, including energetic metabolism, cell cycle, differentiation, apoptosis, and aging. As a result, all sirtuins play critical roles in cellular homeostasis and organism wellness, and their dysregulation has been linked to metabolic, cardiovascular, and neurological diseases. Furthermore, sirtuins have shown dichotomous roles in cancer, acting as context-dependent tumor suppressors or promoters. Given their central role in different cellular processes, sirtuins have attracted increasing research interest aimed at developing both activators and inhibitors. Indeed, sirtuin modulation may have therapeutic effects in many age-related diseases, including diabetes, cardiovascular and neurodegenerative disorders, and cancer. Moreover, isoform selective modulators may increase our knowledge of sirtuin biology and aid to develop better therapies. Through this review, we provide critical insights into sirtuin pharmacology and illustrate their enzymatic activities and biological functions. Furthermore, we outline the most relevant sirtuin modulators in terms of their modes of action, structure-activity relationships, pharmacological effects, and clinical applications.

Current Trends in Sirtuin Activator and Inhibitor Development.

Sirtuins are NAD+-dependent protein deacylases and key metabolic regulators, coupling the cellular energy state with selective lysine deacylation to regulate many downstream cellular processes. Humans encode seven sirtuin isoforms (Sirt1-7) with diverse subcellular localization and deacylase targets. Sirtuins are considered protective anti-aging proteins since increased sirtuin activity is canonically associated with lifespan extension and decreased activity with developing aging-related diseases. However, sirtuins can also assume detrimental cellular roles where increased activity contributes to pathophysiology. Modulation of sirtuin activity by activators and inhibitors thus holds substantial potential for defining the cellular roles of sirtuins in health and disease and developing therapeutics. Instead of being comprehensive, this review discusses the well-characterized sirtuin activators and inhibitors available to date, particularly those with demonstrated selectivity, potency, and cellular activity. This review also provides recommendations regarding the best-in-class sirtuin activators and inhibitors for practical research as sirtuin modulator discovery and refinement evolve.

Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Palladium-Catalyzed Deuteration of Arylketone Oxime Ethers.

We report herein the development of palladium-catalyzed deacylative deuteration of arylketone oxime ethers. This protocol features excellent functional group tolerance, heterocyclic compatibility, and high deuterium incorporation levels. Regioselective deuteration of some biologically important drugs and natural products are showcased via Friedel-Crafts acylation and subsequent deacylative deuteration. Vicinal meta-C-H bond functionalization (including fluorination, arylation, and alkylation) and para-C-H bond deuteration of electro-rich arenes are realized by using the ketone as both directing group and leaving group, which is distinct from aryl halide in conventional dehalogenative deuteration.

Construction of C-S and C-Se Bonds from Unstrained Ketone Precursors under Photoredox Catalysis.

A mild photoredox catalyzed construction of sulfides, disulfides, selenides, sulfoxides and sulfones from unstrained ketone precursors is introduced. Combination of this deacylative process with SN 2 or coupling reactions provides novel and convenient modular strategies toward unsymmetrical or symmetric disulfides. Reactivity studies favor a bromine radical that initiates a HAT (Hydrogen Atom Transfer) from the aminal intermediate resulting in expulsion of a C-centered radical that is intercepted to make C-S and C-Se bonds. Gram scale reactions, broad substrate scope and tolerance towards various functional groups render this method appealing for future applications in the synthesis of organosulfur and selenium complexes.

S-acylation: an orchestrator of the life cycle and function of membrane proteins.

S-acylation is a covalent post-translational modification of proteins with fatty acids, achieved by enzymatic attachment via a labile thioester bond. This modification allows for dynamic control of protein properties and functions in association with cell membranes. This lipid modification regulates a substantial portion of the human proteome and plays an increasingly recognized role throughout the lifespan of affected proteins. Recent technical advancements have propelled the S-acylation field into a 'molecular era', unveiling new insights into its mechanistic intricacies and far-reaching implications. With a striking increase in the number of studies on this modification, new concepts are indeed emerging on the roles of S-acylation in specific cell biology processes and features. After a brief overview of the enzymes involved in S-acylation, this viewpoint focuses on the importance of S-acylation in the homeostasis, function, and coordination of integral membrane proteins. In particular, we put forward the hypotheses that S-acylation is a gatekeeper of membrane protein folding and turnover and a regulator of the formation and dynamics of membrane contact sites.

The Covalent Linking of Organophosphorus Heterocycles to Date Palm Wood-Derived Lignin: Hunting for New Materials with Flame-Retardant Potential.

Environmentally acceptable and renewably sourced flame retardants are in demand. Recent studies have shown that the incorporation of the biopolymer lignin into a polymer can improve its ability to form a char layer upon heating to a high temperature. Char layer formation is a central component of flame-retardant activity. The covalent modification of lignin is an established technique that is being applied to the development of potential flame retardants. In this study, four novel modified lignins were prepared, and their char-forming abilities were assessed using thermogravimetric analysis. The lignin was obtained from date palm wood using a butanosolv pretreatment. The removal of the majority of the ester groups from this heavily acylated lignin was achieved via alkaline hydrolysis. The subsequent modification of the lignin involved the incorporation of an azide functional group and copper-catalysed azide-alkyne cycloaddition reactions. These reactions enabled novel organophosphorus heterocycles to be linked to the lignin. Our preliminary results suggest that the modified lignins had improved char-forming activity compared to the controls. 31P and HSQC NMR and small-molecule X-ray crystallography were used to analyse the prepared compounds and lignins.

The role of structural biology in the design of sirtuin activators.

Sirtuins are NAD+-dependent protein lysine deacylases and mono-ADP-ribosylases whose activity regulates different pathways, including DNA damage repair, cell survival and metabolism, reactive oxygen species (ROS) detoxification, inflammation, cardiac function, and neuronal signaling. Considering the beneficial effects of specific sirtuin isoforms on health and lifespan, the past two decades have seen a mounting interest in the development of sirtuin activators. The availability of enzyme-activator co-crystal structures has proven significant throughout the years for elucidating the mechanisms of action of activators and designing more potent and selective molecules. In this review, we highlight the most interesting examples of sirtuin activators and provide comprehensive coverage of the role that structural biology played in their discovery and characterization.

Deacylative Thiolation by Redox-Neutral Aromatization-Driven C-C Fragmentation of Ketones.

Herein we report the development of deacylative thiolation of diverse methyl ketones. The reaction is redox-neutral, and heavy-metal-free, which provides a new way to introduce thioether groups site-specifically to unactivated aliphatic positions. It also features excellent functional group tolerance and broad substrate scope, thus allowing late-stage derivatization. The process benefits from efficient condensation between the activation reagent and ketone substrates, which triggers aromatization-driven C-C fragmentation and rapid radical coupling with thiosulfonates. Experimental and computational mechanistic studies suggest the involvement of a radical chain pathway.

Unexpected Decarbonylation of Acylethynylpyrroles under the Action of Cyanomethyl Carbanion: A Robust Access to Ethynylpyrroles.

It has been found that the addition of CH2CN- anion to the carbonyl group of acylethynylpyrroles, generated from acetonitrile and t-BuOK, results in the formation of acetylenic alcohols, which undergo unexpectedly easy (room temperature) decomposition to ethynylpyrroles and cyanomethylphenylketones (retro-Favorsky reaction). This finding allows a robust synthesis of ethynylpyrroles in up to 95% yields to be developed. Since acylethynylpyrroles became available, the strategy thus found makes ethynylpyrroles more accessible than earlier. The quantum-chemical calculations (B2PLYP/6-311G**//B3LYP/6-311G**+C-PCM/acetonitrile) confirm the thermodynamic preference of the decomposition of the intermediate acetylenic alcohols to free ethynylpyrroles rather than their potassium derivatives.

Enantioselective bio-deacylation of arylalkyl acetates using tertiary amines as additive under promiscuous conditions.

Herein, we describe the impact of the introduction of tertiary amines as additive during the enzymatic kinetic resolution via deacylation of some arylalkyl acetates 1a-8a under promiscuous conditions. Two CAL-B preparations were examined: Novozym®435 and CHIRAZYME® L-2, c.-f. C2, lyo. The influence of the introduction of seven amines is checked: Triethylamine (Et3N), Pyridine, 4-Dimethylaminopyridine (4-DMAP), 1,4-Diazabicyclo[2.2.2]octane (DABCO), Cinchonine, Cinchonidine, Quinine and Quinidine; and that in two organic solvent: diisopropylether (DIPE) and tertiobutylmethyl ether (TBME). Among the examined amines, the use of DABCO as additive recorded the best results in terms of reactivity and selectivity. It was to be highlighted that this additive was reported as an enzyme activator for the first time in the enzymatic kinetic resolution via hydrolysis of racemic acetates under non-aqueous conditions. Both CAL-B preparations showed the same behavior in the presence of the chosen additives during the biodeacylation of the resolved acetates. No direct correlation between the pKa values of several used additives and the CAL-B activation rates has been revealed. An ideal enzymatic kinetic resolution was recorded with the acetate 4a (Conv 50% and E > >200).

Fluorinated and Non-Fluorinated 1,4-Diarylpyrazoles via MnO2-Mediated Mechanochemical Deacylative Oxidation of 5-Acylpyrazolines.

A solvent-free two-step synthesis of polyfunctionalized pyrazoles under ball-milling mechanochemical conditions was developed. The protocol comprises (3 + 2)-cycloaddition of in situ generated nitrile imines and chalcones, followed by oxidation of the initially formed 5-acylpyrazolines with activated MnO2. The second step proceeds via an exclusive deacylative pathway, to give a series of 1,4-diarylpyrazoles functionalized with a fluorinated (CF3) or non-fluorinated (Ph, COOEt, Ac) substituent at C(3) of the heterocyclic ring. In contrast, MnO2-mediated oxidation of a model isomeric 4-acylpyrazoline proceeded with low chemoselectivity, leading to fully substituted pyrazole as a major product formed via dehydrogenative aromatization. The presented approach extends the scope of the known methods carried out in organic solvents and enables the preparation of polyfunctionalized pyrazoles, which are of general interest in medicine and material sciences.

Exploration of high dimensional free energy landscapes by a combination of temperature-accelerated sliced sampling and parallel biasing.

Temperature-accelerated sliced sampling (TASS) is an enhanced sampling method for achieving accelerated and controlled exploration of high-dimensional free energy landscapes in molecular dynamics simulations. With the aid of umbrella bias potentials, the TASS method realizes a controlled exploration and divide-and-conquer strategy for computing high-dimensional free energy surfaces. In TASS, diffusion of the system in the collective variable (CV) space is enhanced with the help of metadynamics bias and elevated-temperature of the auxiliary degrees of freedom (DOF) that are coupled to the CVs. Usually, a low-dimensional metadynamics bias is applied in TASS. In order to further improve the performance of TASS, we propose here to use a highdimensional metadynamics bias, in the same form as in a parallel bias metadynamics scheme. Here, a modified reweighting scheme, in combination with artificial neural network is used for computing unbiased probability distribution of CVs and projections of high-dimensional free energy surfaces. We first validate the accuracy and efficiency of our method in computing the four-dimensional free energy landscape for alanine tripeptide in vacuo. Subsequently, we employ the approach to calculate the eight-dimensional free energy landscape of alanine pentapeptide in vacuo. Finally, the method is applied to a more realistic problem wherein we compute the broad four-dimensional free energy surface corresponding to the deacylation of a drug molecule which is covalently complexed with a ß-lactamase enzyme. We demonstrate that using parallel bias in TASS improves the efficiency of exploration of high-dimensional free energy landscapes.

Sirtuin modulators: past, present, and future perspectives.

Sirtuins are NAD+-dependent protein lysine deacylase and mono-ADP ribosylases present in both prokaryotes and eukaryotes. The sirtuin family comprises seven isoforms in mammals, each possessing different subcellular localization and biological functions. Sirtuins have received increasing attention in the past two decades given their pivotal functions in a variety of biological contexts, including cytodifferentiation, transcriptional regulation, cell cycle progression, apoptosis, inflammation, metabolism, neurological and cardiovascular physiology and cancer. Consequently, modulation of sirtuin activity has been regarded as a promising therapeutic option for many pathologies. In this review, we provide an up-to-date overview of sirtuin biology and pharmacology. We examine the main features of the most relevant inhibitors and activators, analyzing their structure-activity relationships, applications in biology, and therapeutic potential.

The Zinc-dependent HDACs: Non-histone Substrates and Catalytic Deacylation Beyond Deacetylation.

Protein lysine side chain N(epsilon)-acylation and -deacylation play an important regulatory role in both epigenetic and non-epigenetic processes via a structural and functional regulation of histone and non-histone proteins. The enzymes catalyzing deacylation were traditionally termed as the histone deacetylases (HDACs) since histone proteins were the first substrates identified and the deacetylation was the first type of deacylation identified. However, it has now been known that, besides the seven sirtuins (i.e. SIRT1-7, the ß-nicotinamide adenine dinucleotide (ß-NAD+)-dependent class III HDACs), several of the other eleven members of the mammalian HDAC family (i.e. HDAC1-11, the zinc-dependent classes I, II, and IV HDACs) have been found to also accept nonhistone proteins as native substrates and to also catalyze the removal of the acyl groups other than acetyl, such as formyl, crotonyl, and myristoyl. In this mini-review, I will first integrate the current literature coverage on the non-histone substrates and the catalytic deacylation (beyond deacetylation) of the zinc-dependent HDACs, which will be followed by an address on the functional interrogation and pharmacological exploitation (inhibitor design) of the zinc-dependent HDAC-catalyzed deacylation (beyond deacetylation).

Toward an Orthogonal Protein Lysine Acylation and Deacylation System.

Lysine acetylation is one of the most basic molecular mechanisms to mediate protein functions in living organisms, and its abnormal regulation has been linked to many diseases. The drug development associated to this process is of great significance but severely hindered by the complex interplay of lysine acetylation and deacetylation in thousands of proteins, and we reasoned that targeting a specific protein acetylation or deacetylation event instead of the related enzymes should be a feasible solution to this issue. Toward this goal, we devised an orthogonal lysine acylation and deacylation (OKAD) system, which potentially could precisely dissect the biological consequence of an individual acetylation or deacetylation event in living cells. The system includes a genetically encoded acylated lysine (PhOAcK) that is not a substrate of endogenous deacetylases, and an evolved sirtuin (CobB2/CobB3) that displays PhOAcK deacylase activities as well as reduced deacetylase activities. We believe the strategy introduced here holds potential for future in-depth biological applications.

High-Light-Induced Stress Activates Lipid Deacylation at the Sn-2 Position in the Cyanobacterium Synechocystis Sp. PCC 6803.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) produce free fatty acids (FFAs) because the FFAs generated by deacylation of membrane lipids cannot be recycled. An engineered Aas-deficient mutant of Synechocystis sp. PCC 6803 grew normally under low-light (LL) conditions (50 µmol photons m-2 s-1) but was unable to sustain growth under high-light (HL) conditions (400 µmol photons m-2 s-1), revealing a crucial role of Aas in survival under the HL conditions. Several-times larger amounts of FFAs were produced by HL-exposed cultures than LL-grown cultures. Palmitic acid accounted for ∼85% of total FFAs in HL-exposed cultures, while C18 fatty acids (FAs) constituted ∼80% of the FFAs in LL-grown cultures. Since C16 FAs are esterified to the sn-2 position of lipids in the Synechocystis species, it was deduced that HL irradiation activated deacylation of lipids at the sn-2 position. Heterologous expression of FarB, the FFA exporter protein of Neisseria lactamica, prevented intracellular FFA accumulation and rescued the growth defect of the mutant under HL, indicating that intracellular FFA was the cause of growth inhibition. FarB expression also decreased the 'per-cell' yield of FFA under HL by 90% and decreased the proportion of palmitic acid to ∼15% of total FFA. These results indicated that the HL-induced lipid deacylation is triggered not by strong light per se but by HL-induced damage to the cells. It was deduced that there is a positive feedback loop between HL-induced damage and lipid deacylation, which is lethal unless FFA accumulation is prevented by Aas.

HDAC11: a multifaceted histone deacetylase with proficient fatty deacylase activity and its roles in physiological processes.

The histone deacetylases (HDACs) family of enzymes possess deacylase activity for histone and nonhistone proteins; HDAC11 is the latest discovered HDAC and the only member of class IV. Besides its shared HDAC family catalytical activity, recent studies underline HDAC11 as a multifaceted enzyme with a very efficient long-chain fatty acid deacylase activity, which has open a whole new field of action for this protein. Here, we summarize the importance of HDAC11 in a vast array of cellular pathways, which has been recently highlighted by discoveries about its subcellular localization, biochemical features, and its regulation by microRNAs and long noncoding RNAs, as well as its new targets and interactors. Additionally, we discuss the recent work showing the consequences of HDAC11 dysregulation in brain, skeletal muscle, and adipose tissue, and during regeneration in response to kidney, skeletal muscle, and vascular injuries, underscoring HDAC11 as an emerging hub protein with physiological functions that are much more extensive than previously thought, and with important implications in human diseases.

Notum deacylates octanoylated ghrelin.

OBJECTIVES: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. METHODS: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. RESULTS: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. CONCLUSIONS: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.

Utilization of Unsymmetric Diaryliodonium Salts in α-Arylation of α-Fluoroacetoacetamides.

The use of unsymmetric diaryliodonium salts as a versatile class of arylating agents has been demonstrated by developing a novel strategy to quickly access α-arylated α-fluoroacetoacetamides. The protocol provides a convenient metal-free method for the α-arylation of a diverse class of fluorinated acetoacetamides, and the products are obtained in good yields. The strategy, upon use of electron-deficient diaryliodonium salts as an arylating agent, provides α-fluoroacetamides through a spontaneous arylation/deacylation cascade.